Cystic Fibrosis (CF) is the most common genetic disease among the Caucasians. The affected individuals suffer from insufficient water content and excess mucus in the airway epithelium, due to the lack of cAMP- dependent C1 transport activity of the epithelial cells. Since CFTR gene has been identified to encode for the cAMP dependent C1 channel missing in the CF individuals, gene therapy in the CF airway epithelial cells has become an attractive possibility. Retroviral vectors are unsuitable for this purpose because most of the epithelial cells are non-dividing. Adenovirus has shown a great promise as a vector for gene therapy in the airway, but it is a non-integration vector which necessitates repeated treatment. The immunogenicity of the viral vectors would be a serious problem for patients receiving repeated treatment. Adeno-associated virus (AAV) on the other hand is an integration vector which efficiently integrates its genome into a specific site on human chromosome 19. However, the virus is just as immunogenic as the adenovirus. Furthermore, the integrated viral vector may be re-activated from the host genome and becomes airborn virus when the host cells are infected with wild type AAV and a helper virus, thus presenting a potential environmental health hazard. We propose to overcome these problems by using full length CFTR cDNA plus appropriate regulatory sequence, flanked with the terminal repeat sequence of AAV. This vector sequence (>5Kb) would be too large to be packaged into AAV virion, thus presenting little change of becoming intact virus to re-enter the environment. The long vector sequence will be delivered to the airway epithelial cells with cationic liposome reagents recently developed in our lab. These reagents have been proven efficient for DNA mediated transfection in both dividing and nondividing cells. Thus our approach would combine the advantages of two vector systems, i.e. the ability to transfect non-dividing cells and the ability to integrate into the host genome in a site-specific manner for a safe and long-term expression of the transgene. Furthermore, the new hybrid vector system is nonimmunogenic which would be suitable for repeated treatment if necessary. Specifically, we would like to achieve the following aims: (a) to test the efficiency, integration and toxicity of the hybrid vector system in cultured human epithelial cells using reporter genes, (b) to test the efficiency, integration and toxicity of the hybrid vector system containing CFTR gene in cultured CF epithelial cells which will be examined for the restored CAMP dependent C1 channel activity, and (c) to test in animal models if CFTR gene can be delivered, integrated and expressed in the airway epithelial cells using the vector system previously optimized. These studies should pave the way for human trials of CF gene therapy.